This project will continue our work related to the role of the vascular endothelial cell in the vascular pathobiology of sickle cell disease. The focus of this project will be on our central hypothesis, that thrombospondin (TSP) is the major plasma adhesogen for sickle RBC interaction with the vascular endothelium. Specific Aim 1 is to define the role of TSP and its receptors in mediating sickle RBC adhesion to endothelium. We will address the binding specificity of RBC CD36, and the proposed differences in mechanisms of RBC binding to soluble versus immobilized TSP. We will measure the binding of TSP to endothelial cells, hypothesizing that this involves cooperative participation of multiple receptors, with a major role for CD36 on microvascular endothelial cells. The relative adhesogenicity of TSP versus other proteins in plasma will be tested, and we will search for a TSP/fibrinogen complex as an adhesogenic form of TSP. Also, the influence of environmental factors that modulate endothelium on TSP-mediated RBC adhesion will be tested. Specific Aim 2 will define the role of platelets in TSP-mediated sickle RBC adhesion to endothelium. We hypothesize that the presence of platelets, both resting and activated, has an augmenting effect on sickle RBC adhesion and that multiple platelet surface receptors, especially CD36, participate in this effect. Specific Aim 3 will define the mechanisms of TSP-mediated sickle RBC adhesion that are relevant during flow. In this Aim the major conclusion from Specific Aims 1 and 2 will be directly tested on a flowing system, and promising anti-adhesive therapeutics will be tested. These studies will utilize classical binding and adhesion assays, cultured cell lines (including molecularly engineered endothelial cells), and multiple strategies. These studies will rigorously test our hypotheses regarding the participation of TSP and CD36 in mediating sickle RBC adhesion to endothelium.